In a First, Researchers Use CRISPR to Study Gene Function in Human Embryos

In the first reported first use of CRISPR/Cas9 genome editing to study gene function in developing human preimplantation embryos, researchers from the U.K. and South Korea have applied the technology to target the POU5F1 gene, which encodes the pluripotency transcription factor OCT4 during embryogenesis.

“If we knew the key genes that embryos need to develop successfully, we could improve in vitro fertilization (IVF) treatments and understand some causes of pregnancy failure,” said Kathy Niakan, Ph.D., from the Francis Crick Institute, who led the research. “Now that we have demonstrated an efficient way of doing this, we hope that other scientists will use it to find out the roles of other genes. It may take many years to achieve such an understanding. Our study is just the first step.”

Dr. Niakan and colleagues at the Francis Crick Institute, Cambridge University, Oxford University, the Wellcome Trust Sanger Institute, Bourn Hall Clinic, and Seoul National University, published their findings today in Nature, in a paper entitled "Genome Editing Reveals a Role for OCT4 in Human Embryogenesis.”

The reasons for studying OCT4 were manifold, Dr. Niakan told a press briefing. First, previous work “…had shown that OCT4 is first detectable in eight-cell embryos, and that OCT4 is really unique…in the way it is localized around six days after fertilization and that unique localization is correlated with the perfect time to derive human embryonic stem cells. That suggests that OCT4 may have a role in embryonic stem cells, and we know from the work of others…that if you decrease the level of OCT4 in exiting human embryonic stem cells, that would push the stem cells to exit this pluripotent embryonic state…” and enter differentiation. So that again suggests that OCT4 has an importance in pluripotency and embryonic stem cells.”

Because OCT4 is believed to play a key role in stem cell biology, the findings could provide new insights into the generation and use of pluripotent stem cells. “We have the technology to create and use pluripotent stem cells, which is undoubtedly a fantastic achievement, but we still don't understand exactly how these cells work,” acknowledged James Turner, Ph.D., co-author of the study, from the Francis Crick Institute. The expertise in Dr. Turner’s laboratory was critical to designing the guides and CRISPR strategies used in the published work. “Learning more about how different genes cause cells to become and remain pluripotent will help us to produce and use stem cells more reliably.”

Another reason for choosing OCT4 was to determine whether the protein played different roles in human and mouse embryogenesis, Dr. Niakan suggested. Previous research had already shown that the timing of implantation is different between the two species, for example, and it is known that OCT4 has an important role in mouse embryo development. “… work in model organisms like the mouse suggested that there would be a very obvious effect if we inactivated OCT4 expression.”

The researchers first optimized the CRISPR/Cas9 editing technique using mouse embryos and human embryonic stem cells. “We spent over a year optimizing the various different methodologies, optimizing the different guides that could be used to target OCT4, optimizing the different microinjection techniques, before we ever attempted to use this technology in human embryos,” Dr. Niakan continued. “We wanted to be absolutely sure that all of our different methods were right and to minimize the number of human embryos we used to understand the function and the importance of this gene.”

With this development work complete, the team then applied the refined CRISPR/Cas9 genome-editing technology to target POU5F1 in 41 previously frozen human zygotes that had been donated for research by couples who had undergone IVF. The study was carried out under license from and oversight by the U.K.’s Human Fertilisation and Embryology Authority (HFEA), which oversees infertility treatment and research.

The findings confirmed that preventing OCT4 production in these very early embryos had critical consequences. “When we inactivated OCT4 what we found was that there were fewer embryos that could develop to the blastocyst stage,” Dr. Niakan told the press briefing. “So that tells us that OCT4 is really important for human embryos to develop and reach this important stage of embryogenesis…it's absolutely critical for that.”

Another key finding was that preventing OCT4 production has very different effects on mouse and human embryo development. “Unexpectedly, our data suggest that OCT4 may be required earlier in human development than in mice, for instance, during the cleavage or morula stages, when OCT4 expression is initiated,” the researchers write.

Dr. Niakan commented: “The really fascinating thing for us is that when we look at a control blastocyst compared to one in which we have inactivated OCT4, we see that there is an effect on those 20 so-called epiblast progenitor cells…the precursors of what will eventually become the embryo. That’s different to model organisms, such as the mouse. So we would have never gained this insight if we had not studied the function of this gene in human embryos, and that tells us that there may be important differences in the pluripotency, potentially between these species.

“The other important thing that we found is that we see not only an effect on those 20 cells, but also on the precursor cells of the placenta, and that is completely different to the mouse,” Dr. Niakan added.

The effects of genome editing on placenta precursor cells wasn’t expected, she admitted. “That’s not predicted anywhere in the literature, and so that suggests that there are more essential roles of OCT4. This is an area that we are really interested to understand, and we will be spending quite a lot of time on this in the future, to uncover what this role might be.”

The researchers more fully describe the consequences of OCT4 loss on embryo gene expression, in their published Nature paper. “We were surprised to see just how crucial this gene is for human embryo development, but we need to continue our work to confirm its role,” commented Norah Fogarty, Ph.D., from the Francis Crick Institute, who is lead author of the paper.

“This proof of principle lays out a framework for future investigations that could transform our understanding of human biology, thereby leading to improvements in the establishment and therapeutic use of stem cells and in IVF treatments,” the authors conclude.

Successful IVF treatment depends on the ability to provide culture systems that represent the best environment for healthy embryo development, commented Kay Elder, M.D., Ph.D., study co-author from Bourn Hall Clinic. “Many embryos arrest in culture, or fail to continue developing after implantation; this research will significantly help treatment for infertile couples by helping us to identify the factors that are essential for ensuring that human embryos can develop into healthy babies.”

Sir Paul Nurse, director of the Francis Crick Institute, added: “The study has been carried out with full regulatory oversight and offers new knowledge of the biological processes at work in the first five or six days of a human embryo's healthy development. Dr. Niakan and colleagues are providing new understanding of the genes responsible for a crucial change when groups of cells in the very early embryo first become organized and set on different paths of development. The processes at work in these embryonic cells will be of interest in many areas of stem cell biology and medicine."

Ludovic Vallier, Ph.D., study co-author from the Wellcome Trust Sanger Institute and the Wellcome-MRC Cambridge Stem Cell Institute, stressed the importance of the study as a step in understanding human embryo development: “The acquisition of this knowledge will be essential to develop new treatments against developmental disorders and could also help understand adult diseases such as diabetes that may originate during the early stage of life. Thus, this research will open new fields of opportunity for basic and translational applications.”

Dr. Vallier’s laboratory played a critical role in testing the genome-editing technologies in human embryonic stem cells and optimizing the genome-editing technology in vitro.

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